1. Field of the Invention
The present invention relates to a method and a device for initiation of cell growth and for cultivation of cells in high densities. The cells to be cultivated are located in hollow-filament membranes and are brought alternately into a liquid nutrient medium and a gas phase.
2. Description of the Related Art
Mammalian cell cultivation for the synthesis of biopharmaceutical drugs is performed mainly in stirred reactors. Heretofore, airlift reactors have been used less frequently and hollow-fiber reactors very rarely for servicing the market with drugs based on mammalian cells. To improve the volumetric product yields in stirred reactors, the cell density and the effective production time of the cells are increased by optimizing the methods and using nutritional regimens specific to the cell lines in fed batch methods. The production technology is laid out in bioreactor trains containing three to four stirred reactors, each with a volumetric capacity of approximately five times that of the preceding bioreactor. The largest available stirred reactor for cultivation of mammalian cells currently has a volumetric capacity of 20,000 liters. Fed-batch processes in stirred reactors are robust, can be scaled up to the above volumes and long ago were accepted by the authorities for drug synthesis. Disadvantages are the long dwell times of the products in the culture chamber, the need for separation of cells from the harvest supernatant, the cleaning and sterilization expenses incurred during multiple use and the high investment and operating expenses for plants equipped with this technology.
For proteins, such as human factor VII, a protein of the clotting cascade, which are susceptible to degradation and thus impose a short dwell time in the bioreactor during synthesis, there have been developed devices and systems that permit perfusion of the culture chamber and thus continuous operation of the stirred reactors. For this purpose, efficient cell retention with continuous media feed and product harvesting is necessary. Spin filters are used here in the interior chamber of the stirred reactor, while support materials in the form of fluidized or stationary beds are used in the traps, where the production cells can adhere to surfaces. The continuous mode of operation of stirred reactors can also be achieved via external cell-retention systems, such as cell sedimentation, continuous cell centrifugation or ultrasonic cell collection. Advantages of the continuous mode of operation are short product dwell times in the bioreactor, constant product quality during synthesis, increase of the volumetric productivity and greater flexibility of batch volume as a function of the cultivation time to be defined. Disadvantages are contamination of the harvest with residual cells, the cleaning and sterilization expenses incurred for multiple use and the high investment and operating costs for the corresponding plants.
Besides the hollow-fiber bioreactors of ACUSYST® X Cell Generation, which have proved effective for the synthesis of biopharmaceuticals, other reactor systems are available in which all components coming in contact with the cell culture are designed as disposable components. Thus they can be discarded once they have been used to synthesize a batch. Expensive cleaning and sterilization procedures are not required. Commercially available systems of this type are membrane-based systems such as Cell-Pharm®, Cellmax®, Technomouse®, CELLine®, miniPERM® or OptiCell®. Membrane methods have several advantages. In perfusion operation they can achieve very high cell densities (107 to 108 cells/ml)—by virtue of a large membrane surface per unit volume. Moreover, the cells are protected by the membranes from shearing forces. In principle, they are designed for one-time use, so that cleaning and sterilization after use are not necessary. In the art of disposable bioreactors, the wave bioreactor has also proved effective heretofore in the trial phase for the synthesis of biopharmaceuticals. In the system, the cells are cultivated in a bag system, which is systematically agitated in order to improve intimate mixing. One advantage of this reactor technology is the one-time usability of the culture system. Disadvantages are the low achievable densities and the limited scale-up capability.
In all cited methods and devices, uniform nutrient supply and in particular oxygen supply at high cell densities is problematic. Neither the attempt to solve this problem via complex process steps involving pressurization (1989, U.S. Pat. No. 4,804,628 A) nor the direct introduction of oxygen into the cell culture chamber via a further membrane system (1986, DE 2431450 A1 and 1995, DE 4230194 A1) led to culture systems whose scale could be increased as desired and in which the cells could be uniformly supplied. In hollow-fiber bioreactors, in which the cells are cultivated between the hollow fibers and the nutrients are transported in the lumen of the fibers, scale-up is limited by the length of the hollow fibers. However, the length of the hollow fibers is limited by consumption of the oxygen from the hollow fibers. Thereby scale-up is possible only by the use of parallel units. In practice, however, this leads to unprofitable processes. In other words, the scale-up capability of the hollow-fiber reactors is defeated by the lack of adequate homogeneous supply of the cells with fresh gas and liquid nutrient components.
In WO 03/064586 A2, it was proposed that cells be cultivated in high density in compartments, the dimension of which compartments is not to exceed 5 mm in length. The interior chamber of the compartments forms a culture chamber, which is partitioned from the supply chamber by a semipermeable element. The cells are retained in the compartments, and oxygen exchange takes place via hollow-fiber membranes. Supply of the cells with nutrients and with oxygen is ensured by means of a variably adjustable mixture of gas and cell-culture media. Although the culture device and the method solve the problem of nutrient and oxygen supply and guarantee scale-up capability, the method described in WO 03/064586 A2 suffers from a disadvantage in that cells of high density must be introduced into the compartments. To overcome this disadvantage, it was proposed in WO 03/102123 A2 that biodegradable gels be used to reduce the inoculation density at the beginning of cell cultivation.
A liquid-gas-phase exposure bioreactor has been developed in principle by the Zellwerk® Co. and is being sold by the Sartorius® Co. In this bioreactor, the cells that adhere to surfaces are immobilized on disks of carrier material. The disks are disposed in series on a shaft, and are rotated in a cylinder that is half-filled with medium and half-filled with gas. An advantage of this arrangement is the cyclic exposure of these cells to both phases. Disadvantages are the limitation of the system and method to adhering cells, the presence of detached cells in the harvest fluid and the limitation of scale-up capability.